Techniques in molecular biology and molecular medicine often rely on analysis of single biological molecules. Such techniques include DNA and RNA sequencing, polymorphism detection, the detection of proteins of interest, the detection of protein-nucleic acid complexes, and many others. The high sensitivity, high throughput and low reagent costs involved in single molecule analysis make this type of analysis an increasingly attractive approach for a variety of detection and analysis problems in molecular medicine, from low cost genomics to high sensitivity marker analysis.
For example, single molecule DNA sequencing is useful for the analysis of large sets of related DNAs, such as those that occur in a genome. In some sequencing methods, a polymerase reaction is isolated within an array of extremely small (typically optically confined) observation volumes that permit observation of the enzymatic action of individual polymerases in each reaction/observation volume of the array, while the polymerase copies a template nucleic acid. Nucleotide incorporation events are individually detected, ultimately providing the sequence of the template molecule. This approach dramatically increases throughput of sequencing systems while also dramatically reducing reagent consumption costs, making personalized genomics increasingly feasible.
The small observation volumes often used for single molecule nucleic acid sequencing and other analysis methods are typically provided by immobilizing or otherwise localizing the polymerase (or other) enzyme within a reaction region, which can include an array of extremely smalls wells, such as in an array of Zero Mode Waveguides (ZMWs), and delivering a template, primers, etc., to the wells. One difficulty in performing single molecule analyses occurs in efficiently loading the reaction/observation region of single molecule analysis devices with the molecules of interest (e.g., template or other analyte and/or enzyme or any other associated molecules). Methods of loading that rely on diffusion often require large concentrations of sample in order to load a particular density of reaction regions in a given period of time. It would be desirable to develop methods and compositions for increasing the speed with which molecules are loaded into the reaction/observation regions and thus require lower concentrations of initial sample. Increased efficiency in loading would thus reduce cost and time in terms of sample volumes required and would also simultaneously increase the throughput of such systems. The present disclosure provides these and other features that will be apparent upon complete review of the following.